Name: impedance-based real-time measurement of cancer cell migration and invasion
Uploaded: 2020-04-02T15:00:00
Description: Watch this Scientific Journal Video about Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion at JoVE.com

We thank Olivia Funk for her technical assistance with the real-time cell analysis system data. We also thank the Medical Writing Center at Children&#8217;s Mercy Kansas City for editing this manuscript. This work was supported by Tom Keaveny Endowed Fund for Pediatric Cancer Research (to TP) and by Children&#8217;s Mercy Hospital Midwest Cancer Alliance Partner Advisory Board funding (to TP).

NOTE: All cell culture materials need to be sterile and the entire experiment must be performed in a biosafety cabinet under sterile conditions.
1. Culture and Electroporation of the U-118MG Glioblastoma Cell Line
<ol>
	<li>Culture the U-118MG cell line in 5% fetal bovine serum (FBS) containing Dulbecco&#8217;s Modified Eagle Medium (DMEM) (culture medium) and Maintain at 37 &#176;C in a humid atmosphere containing 5% CO2 incubator (culture conditions).</li>
	<li>...

<ol>
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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+proteins+transduce+FGF+signaling+to+promote+lens+fiber+cell+elongation.">Crk proteins transduce FGF signaling to promote lens fiber cell elongation.</a> Elife. 7, (2018).</li><li>Fathers, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+adaptor+proteins+act+as+key+signaling+integrators+for+breast+tumorigenesis.">Crk adaptor proteins act as key signaling integrators for breast tumorigenesis.</a> Breast Cancer Research. 14 (3), 74 (2012).</li><li>Koptyra, M., Park, T. J., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+and+CrkL+are+required+for+cell+transformation+by+v-fos+and+v-ras.">Crk and CrkL are required for cell transformation by v-fos and v-ras.</a> Molecular Carcinogenesis. 55 (1), 97-104 (2016).</li><li>Lamorte, L., Royal, I., Naujokas, M., Park, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+adapter+proteins+promote+an+epithelial-mesenchymal-like+transition+and+are+required+for+HGF-mediated+cell+spreading+and+breakdown+of+epithelial+adherens+junctions.">Crk adapter proteins promote an epithelial-mesenchymal-like transition and are required for HGF-mediated cell spreading and breakdown of epithelial adherens junctions.</a> Molecular Biology of the Cell. 13 (5), 1449-1461 (2002).</li><li>Park, T. J., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+roles+of+Crk+and+CrkL+in+fibroblast+structure+and+motility.">Essential roles of Crk and CrkL in fibroblast structure and motility.</a> Oncogene. 33 (43), 5121-5132 (2014).</li><li>Rodrigues, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrkI+and+CrkII+function+as+key+signaling+integrators+for+migration+and+invasion+of+cancer+cells.">CrkI and CrkII function as key signaling integrators for migration and invasion of cancer cells.</a> Molecular Cancer Research. 3 (4), 183-194 (2005).</li><li>Feller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+family+adaptors-signalling+complex+formation+and+biological+roles.">Crk family adaptors-signalling complex formation and biological roles.</a> Oncogene. 20 (44), 6348-6371 (2001).</li><li>Park, T. J., Boyd, K., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+and+craniofacial+defects+in+Crk-null+mice.">Cardiovascular and craniofacial defects in Crk-null mice.</a> Molecular and Cellular Biology. 26 (16), 6272-6282 (2006).</li><li>Park, T. J., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+and+Crk-like+play+essential+overlapping+roles+downstream+of+disabled-1+in+the+Reelin+pathway.">Crk and Crk-like play essential overlapping roles downstream of disabled-1 in the Reelin pathway.</a> Journal of Neuroscience. 28 (50), 13551-13562 (2008).</li><li>Hallock, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dok-7+regulates+neuromuscular+synapse+formation+by+recruiting+Crk+and+Crk-L.">Dok-7 regulates neuromuscular synapse formation by recruiting Crk and Crk-L.</a> Genes &amp; Development. 24 (21), 2451-2461 (2010).</li></ol>

The real-time measurement of cell migration and invasion using the real-time cell analysis system is a simple, quick, and continuous monitoring process with multiple, significant advantages over the traditional methods that provide data at a single time point. As with the traditional methods, experimental conditions must be optimized for each cell line for the real-time cell analysis system, because each cell line may be different in terms of its adhesion to the substrate, growth, cell-to-cell contacts, and migratory and...

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining cancer genotypes and phenotypes is critical to understanding and designing effective therapeutic strategies. Decades of cancer research have led to the development and adaptation of different methods to determine cancer genotypes and phenotypes. One of the latest technical developments is real-time measurement of cell migration and invasion based on cell impedance. Cell adhesion to substrates and cell-cell contacts play an important role in cell-to-cell communication and regulation, development, and maintenance of tissues. Abnormalities in cell adhesion lead to the loss of cell-cell contact, degradation of extracellular matrix (ECM), and gain of migratory and invading capabilities by cells, all of which contribute to metastasis of cancer cells to different organs1,2. Various methods are available to determine cell migration (wound healing and Boyden chamber assays) and invasion (Matrigel-Boyden chamber assay)3,4,5. These conventional methods are semiquantitative because cells need to be labeled with a fluorescent dye or other dyes either before or after the experiment to measure cell phenotypes. In addition, mechanical disruptions are needed in some cases for creating a wound for measuring the migration of cells to the wound site. Moreover, these existing methods are time-consuming, labor-intensive, and measure the results at only one time point. In addition, these methods are prone to making inaccurate measurements due to inconsistent handling during the experimental procedure6.
Unlike conventional methods, the real-time cell analysis system measures cell impedance in real-time without requiring pre- or poststaining and mechanical damage of cells. More importantly, the duration of an experiment can be extended so that biological effects can be determined in a time-dependent manner. Executing the experiment is time-efficient and not labor-intensive. Analyzing data is relatively simple and accurate. Compared to other methods, this method is one of the best real-time measurements to measure cell migration and invasion6,7,8,9.
Giaever and Keese were the first to describe the impedance-based measurement of a cell population on the surface of electrodes10. The real-time cell analysis system works on the same principle. The area of each microplate well is approximately 80% covered with an array of gold microelectrodes. When the electrode surface area is occupied by cells due to adherence or spreading of the cells, the electrical impedance changes. This impedance is displayed as the cell index, which is directly proportional to the cells covering the electrode surface area after they penetrate the microporous membrane (the median pore size of this membrane is 8 &#956;m)11.
Crk and CrkL are adaptor proteins containing SH2 and SH3 domains and play important roles in various cellular functions, such as cytoskeleton regulation, cell transformation, proliferation, adhesion, epithelial-mesenchymal transition, migration, invasion, and metastasis by mediating protein-protein interactions in many signaling pathways1,12,13,14,15,16,17,18. Therefore, it is important to determine the Crk/CrkL-dependent migratory and invasive capabilities of cancer cells. Real-time cell analysis was performed to determine the migratory and invasive abilities of glioblastoma cells upon gene knockdown of Crk and CrkL.
This method paper describes detailed measurements of Crk- and CrkL-mediated migration and invasion of human glioblastoma cells.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:1140px;" width="1140">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Biosafety cabinet</td>
			<td style="width:339px;">ThermoFisher Scientific</td>
			<td style="width:235px;">1300 Series Class II, Type A2</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CIM plates</td>
			<td>Cell Analysis Division of Agilent Technologies, Inc</td>
			<td>5665825001</td>
			<td>Cell invasion and migration plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Crk siRNA</td>
			<td>Dharmacon</td>
			<td>J-010503-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CrkL siRNA</td>
			<td>Ambion</td>
			<td>ID: 3522 and ID: 3524</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#8217;s modified eagle&#8217;s medium (DMEM)</td>
			<td>ATCC</td>
			<td>302002</td>
			<td>Culture medium used for cell culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Gibco</td>
			<td>21-031-CV</td>
			<td>DPBS used to wash the cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heracell VIOS 160i CO2 incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>51030285</td>
			<td>Co2 incubator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Matrigel</td>
			<td>BD Bioscience</td>
			<td>354234</td>
			<td>Extracellular matrix gel</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neon electroporation system</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK5000</td>
			<td>Electroporation system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neon transfection system 10 &#181;L kit</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK1025</td>
			<td>Electroporation kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Non-targeting siRNA</td>
			<td>Dharmacon</td>
			<td>D-001810-01</td>
			<td>siRNA for non targated control</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Odyssey CLx (Imaging system)</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td>Western blot imaging system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RTCA software</td>
			<td>Cell Analysis Division of Agilent Technologies, Inc</td>
			<td></td>
			<td>Instrument used for experiment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Scepter</td>
			<td>Millipore</td>
			<td>C85360</td>
			<td>Handheld automated cell counter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">U-118MG</td>
			<td>ATCC</td>
			<td>ATCC HTB15</td>
			<td>Cell lines used for experiments</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">xCELLigence RTCA DP</td>
			<td>Cell Analysis Division of Agilent Technologies, Inc</td>
			<td>380601050</td>
			<td>Instrument used for experiment</td>
		</tr>
	</tbody>
</table>

impedance-based real-time measurement of cancer cell migration and invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

It has been suggested that Crk and CrkL are important for cell migration and invasion in different cancer cell lines13,17. Although Crk and CrkL proteins are structurally and functionally similar to each other and play essential overlapping functions16,19,20,21, many gene knockdown studies for Crk and CrkL have not clearly addressed whe...

Watch this Scientific Journal Video about Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion at JoVE.com

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Using this protocol, cell migration and invasion can be unveiled under real-time conditions enabling cell kinetics under loss or gain of gene function and drugs to be determined. Unlike other methods, no staining, mechanical cell damage, or fluorescent markers needed. Most importantly, the real-time cell kinetics can be determined in an effective manner.When the U-118MG cell line culture reaches 70 to 80%confluency, wash the cells with PBS before treating the cells with two milliliters of 0.5%trypsin-EDTA. After 30 seconds at 37 degrees Celsius, stop the reaction with 10 milliliters of fresh culture medium and transfer the detached cells to a 15 milliliter conical tube. After counting, collect the cells by centrifugation and resuspend the pellet in a six times 10 to the fifth cells per condition concentration in the appropriate volume of fresh medium.Transfer six times 10 to the fifth cells into one microcentrifuge tube per condition and add 800 microliters of Dulbecco's PBS to each tube. After centrifugation, resuspend each pellet in 60 microliters of resuspension buffer R and six micromolar of the siRNA of interest. Mix each tube with gentle tapping before electroporating 10 microliter aliquots of each cell suspension with three 10-millisecond 1, 350 volt pulses.After each treatment, pool the electroporated cells from each condition in individual five milliliter aliquots of fresh culture medium. Then seed the cells into two 35 millimeter culture dishes per condition and place the cells in the cell culture incubator for three days. Five to six hours before the real-time cell analysis, place the real-time cell analysis system into the cell culture incubator.To set up an invasion assay, use reverse pipetting to place 50 microliters of DMEM supplemented with 0.1 microgram per milliliter of extracellular matrix gel into each well of the upper chamber of a cell invasion plate. Immediately after plating, remove 30 microliters of the extracellular matrix solution from each well and place the plate into the cell culture incubator for four hours. To set up an impedance measurement program, six hours before the measurement, replace the medium in all of the electroporated cell cultures with low serum medium.Under the Layout tab in the associated impedance measurement software, select quadruplicate wells for each biological condition. Under the Schedule tab, set a one-time baseline measurement sweep step with a one-minute interval and set a second step to measure the cell impedance in respective individual cradles for the actual experiment. One hour before the start of the cell impedance measurement, add 160 microliters of medium supplemented with 10%fetal bovine serum as the chemoattractant into the wells of the lower chamber of the cell invasion and migration plate.To measure invasion, assemble the upper chamber containing the extracellular matrix gel coated wells. To measure migration, use uncoated wells in the upper chamber. For either type of experiment, fill the wells in the upper chamber with 50 microliters of low serum medium and place the chambers into the cradle of the system.Click the Message tab in the software to determine whether all of the wells are recognized by the control unit. If the message displays as expected, the plate in the cradle is ready for the experiment. Then place the completely packed plates into the cell culture incubator in the real-time cell analysis system cradle for one hour to acclimate the plate to the cell culture conditions.While the plates are equilibrating, harvest the glioblastoma cells as demonstrated and resuspend the cells from each condition at an eight times 10 to the fifth cells per 800 microliters of low serum medium concentration. Additionally, set aside a three times 10 to the fifth cells in two milliliters of culture medium for seeding in a 35 millimeter dish for downstream Western blot analysis. To measure the baseline pre-migration reading, at the end of the acclimatization, click the cradle Start button.After the baseline measurement has been acquired, transfer the migration and the invasion plates from their respective cradles into a biosafety cabinet. Reverse pipette 100 microliters of cells into quadruplicate upper chamber wells for each biological condition in the appropriate well of the cell invasion and migration plates as programmed in the control unit of the cradle. After seeding, keep the plates in the biosafety cabinet for 30 minutes at room temperature to allow the cells to settle evenly on the plate bottom before transferring the plates to their respective cradles.Click the cradle Start button to begin measuring the cell impedance. To visualize the changes in cell impedance as the cell index in a time-dependent manner during or after completion of the experiment, open the Data Analysis tab. To visualize the data for each of the respective conditions either individually or as averages and/or standard deviations, click the option boxes for average and standard deviation.To export the cell index data to a spreadsheet file, place the cursor in the middle of the data analysis window and right-click. In the dialogue box that appears, select the copy data into list format option and paste the data into an open spreadsheet. To release the experiment, click the Release button in each cradle.Crk adapter protein knockdown decreases Crk1 and Crk2 protein levels by 85%and 86%respectively without inducing Crk-like protein levels. The knockdown of Crk-like reduces Crk-like protein expression by 85%with slight reductions in Crk1 and Crk2 protein levels. Combining both siRNAs reduces Crk1, Crk2, and Crk-like expression by over 80%while neither Vinculin nor alpha-Tubulin expression is affected by any combination of Crk or Crk-like knockdown.Human brain glioblastoma cells migrate along a gradient in response to high serum concentrations reaching the maximum level of migration at 13 hours. In Crk knockdown cells, although the migration is initially delayed, the cells continued to migrate until 23 hours. Crk-like knockdown substantially reduces cell migration with the glioblastoma cell line completely losing their migratory ability upon knockdown of both Crk and Crk-like suggesting that Crk and Crk-like play essential overlapping roles in cancer cell migration.However, when cell invasion is monitored over several days, the Crk knockdown cells reach a similar maximum level of cell invasion compared to control glioblastoma cells at 60 hours with Crk-like cells partially recovering their invasive ability by 90 hours and double Crk-Crk cells demonstrating a reduced ability to invade after 40 hours. The results clearly support the suggestion that cell invasion should be analyzed over the entire period of the experiment. Seeding the right number of cells and avoiding the air bubbles while recording the extracellular matrix for invasion assay and seeding cells are key points for success of this experiment.Following similar procedure but using e-plates, cell adhesion and cell proliferation kinetics can be determined.

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Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...